Registration Dossier
Registration Dossier
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EC number: 947-049-2 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
Reproduction toxicity (OECD TG 422): NOAEL 1301 mg/kg bw/day in males and 1590 mg/kg bw/day in females (read across from Ylang Ylang I)
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- The full read across justification report is attached under "Attached justification".
23 April 2018 READ-ACROSS STUDY / YLANG YLANG III OIL - REPEATED DOSE TOXICITY AND REPRODUCTIVE TOXICITY I&B9W8768R001F0.1
Executive Summary
According to Annex VIII, 8.6 and 8.7 of the REACh Regulation (EC) No 1907/2006, Repeated dose toxicity and Reproductive toxicity is standard information required for the registration of substances manufactured or imported in quantities of ten tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).
A read-across approach appears appropriate to predict the endpoint “Repeated dose toxicity and Reproductive toxicity” for the substance Ylang Ylang III oil because:
The source substance Ylang Ylang Ext /I /II oil and target substance Ylang Ylang III oil are chemically and in composition very similar and therefore they are expected to exhibit similar properties.
The source substance, gives rise to the highest concern regarding the oral repeated dose toxicity endpoint, as the constituents notified for relevant systemic toxicological potential and/or giving an alert in the QSAR toolbox add up to 8.5 – 70.5 % w/w in the source versus 8.42 – 44.1 % w/w in the target UVCB.This suggests a worst-case situation, implying that the toxicity can be read across to the target substance.
An OECD TG 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, is available for the substance Ylang Ylang I, which has a composition very similar to the target substance.
This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the structures and the Repeated dose toxicity/Reproductive toxicity for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter - Reason / purpose for cross-reference:
- read-across source
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Piloerection was noted on a single occasion at the end of lactation for one control female (no. 50), three 7500 ppm females (nos. 62, 68 and 69) and four 15000 ppm females (nos. 73, 76, 79 and 80). For one 15000 ppm female (no. 78) piloerection was noted on three consecutive days during the first week of lactation. These findings where considered incidental and therefore not treatment related.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Description (incidence):
- Female no. 70 (7500 ppm) was euthanized on PND 4 due to total litter loss.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- No treatment-related changes in body weight or body weight gain were observed in males up to 7500 ppm and in females at 2500 ppm.
In males, body weight and body weight gain was significantly decreased for males at 15000 ppm from Day 4 of the pre-mating phase to Day 15 of the mating period. In females, the following effects were observed:
- At 15000 ppm showed a body weight loss Days 1-11 of the pre mating period. At 15000 ppm differences in body weight gain of -6% for males on Day 15 of the mating period and -5% for females on Day 1 of the mating period were noted, compared to controls.
- In the post-coitum phase, body weights for females treated with 7500 ppm and 15000 ppm were lower compared to controls from Day 0 onwards, which was statistically significant on Day 17 (-7%) for 7500 ppm and 15000 ppm and on Day 20 (-10 %) for 7500 ppm only.
- During lactation, bodyweights for females treated with 7500 ppm and 15000 ppm were lower compared to controls. This was statistically significant for females treated with 15000 ppm during the whole lactation period. For females treated with 7500 ppm this was statistically significant on Day 4 of lactation. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- No treatment-related or toxicologically relevant changes in food consumption before or after allowance for body weight were observed in males up to 7500 ppm and for females at 2500 ppm. In males, food consumption was reduced for 15000 ppm males compared to controls on Days 1-8 of the premating period (both absolute and relative) and Days 1-15 of the mating period. Food consumption corrected for body weight was reduced for 7500 ppm and 15000 ppm males compared to control on Days 1-8 of the mating period. In females of the 15000 ppm dose group, food consumption (both absolute and relative) was reduced compared to controls on Days 1-8 of the premating period, Days 0-20 during the post coitum period and Days 1-5 and Days 6-14 of lactation. For 7500 ppm females food consumption (both absolute and relative) was significantly reduced on Days 1-14 during lactation, which was statistically significant on several days.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- In males, a statistically significant increase (1.2 fold) in creatinine for males at 15000 ppm compared to control was noted. These findings are considered to be adverse.
In females, an increase in ALP was noted, which was statistically significant at 15000 ppm (3.8-fold increase). Although female no. 76 had a markedly higher ALP value, all other females at 15000 ppm had a higher ALP value compared to controls as well. Other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- In males, a test item-related microscopic findings were noted in the kidney of the 7500 and 15000 ppm treated males (hyaline droplet accumulation increased basophilia and granular casts). The hyaline droplet accumulation was considered to represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This is a male rat specific protein and therefore the finding is not considered relevant to humans. In females, test item-related microscopic findings were noted in the thymus at 15000 ppm (minimal lymphoid depletion).
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Reproductive function: oestrous cycle:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Minor and incidental effects were observed and therefore considered non-treatment-related.
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Minor and incidental effects were observed on number of implantation sites and fertility index but no dose-response was observed and therefore these findings were considered non-treatment-related.
No effects were observed on mating index and precoital time.
There were 2/10 couples of the control group and 4/10 couples treated at 7500 ppm, that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain the reproductive failure in these couples, except for couple 61/21 at 7500 ppm. Male 21 of this couple showed a disturbed spermatogenesis which was visible by a bilateral moderate degeneration/atrophy of germ cells in the testes together with luminal cell debris in the epididymides. All other males did not show any indications for abnormal spermatogenesis after stage aware evaluation of the testes. There was 1/10 couples treated at 7500 ppm with total litter loss. Histopathology did not reveal changes in the reproductive organs that could explain this although a decrease in mammary gland development (compared to normal lactating/pregnant females) may possibly be related to the litter loss. - Key result
- Dose descriptor:
- NOAEL
- Remarks:
- Reproduction
- Effect level:
- 1 301 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Remarks on result:
- other: Highest dose tested
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- Reproduction
- Effect level:
- 1 590 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Remarks on result:
- other: Highest dose tested
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The clinical signs observed incidentally in treated pups remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment. The viability indices were 99, 98, 87 and 100% in control, 2500 ppm, 7500 ppm and 15000 ppm group respectively.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body weights of male and female pups were lower at 15000 ppm from PND 7–13. The difference from controls was approximately 17% on PND 13.
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- Developmental
- Generation:
- F1
- Effect level:
- 718 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- body weight and weight gain
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- Developmental
- Generation:
- F1
- Effect level:
- 953 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- body weight and weight gain
- Key result
- Reproductive effects observed:
- no
- Conclusions:
- The repeated dose toxicity of Ylang Ylang III oil was read across from Ylang Ylang I. Under the conditions of this study, the NOAEL for reproduction was considered to be 15000 ppm corresponding to 1301 mg/kg bw/day in males and 1590 mg/kg bw/day in females. The developmental NOAEL was considered to be 7500 ppm, corresponding to 718 mg/kg bw/day in males and 953 mg/kg bw/day in females. Based on this result, Ylang Ylang III does not have to be classified for reproductive toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Executive summary:
The repeated dose toxicity of Ylang Ylang III oil was read across from Ylang Ylang I. The reproductive and developmental toxicity potential of Ylang Ylang I was tested under GLP in a combined repeated dose toxicity study with reproduction/developmental toxicity screening test according to OECD TG 422. The experiment was performed by oral administration of the test substance via diet with 5 rats per sex per dose. The tested dietary doses corresponded to 0, 2500, 7500 and 15000 ppm. Males were treated for 28 days (a minimum of two weeks prior to mating and during the mating period). Females that delivered offspring were treated 49-63 days, a minimum of two weeks prior to mating. Females that delivered no healthy offspring were treated for 41 -51 days. The following parameters and endpoints were evaluated in this study for repeated dose toxicity: mortality/ moribundity, clinical signs, functional observations and locomotor activity, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4, gross necropsy findings, organ weights and histopathologic examinations. The following parameters were evaluated in this study for reproduction and development: Mating index, preciodal time, number of implantation sites, fertility index, gestation index and duration, post implantation survival index, litter size, live birth index, viability index, lactation index. The pups were evaluated for clinical signs, body weights, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry (T4) and macroscopic changes.
In the parental animals, test item related changes included a dose dependent increase in liver weights (relative to body weight was significant), at all dose levels in males and in females at 15000 ppm, in the absence of morphological alterations. For females, but not males, an increase in ALP was noted which was statistically significant at 15000 ppm (3.8–fold increase) which in the presence of the increased liver weight was considered adverse. This increase in liver weights (>20% compared to control) were considered adverse in males and females at 15000 ppm. Test item related increase in kidney weights (only relative to body weight was significant) were observed in males and females treated at 15000 ppm. For females the increased kidney weight in the absence of morphological changes was considered non adverse. In males morphological alterations consisted of a combination of increased hyaline droplet accumulation, increased basophilia and granular casts in males treated at 7500 ppm and 15000 ppm. In addition, creatinine levels were statistically significantly increased for males treated with 15000 ppm. The hyaline droplet accumulation was considered to represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein and is not considered relevant to humans risk assessment. No mortality occurred throughout the study. One female was euthanized on PND 4 due to total litter loss. No relevant clinical signs or neurotoxicity were observed. In addition, no treatment related effects on reproductive toxicity were observed up to the highest dose. In the offspring, no developmental toxicity was observed up to 7500 ppm. Body weights of pups of the 15000 ppm dose group were statistically significantly reduced on PND 7 and 13 (approximately 17% lower compared to control on PND 13), which was considered adverse.
Under the conditions of this study, the NOAEL for reproduction was considered to be 15000 ppm corresponding to 1301 mg/kg bw/day in males and 1590 mg/kg bw/day in females. The developmental NOAEL was considered to be 7500 ppm, corresponding to 718 mg/kg bw/day in males and 953 mg/kg bw/day in females. The repeated dose toxicity of Ylang Ylang III oil was read across from Ylang Ylang I. Based on this result, Ylang Ylang III does not have to be classified for reproductive toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05-04-2017 to 31-07-2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Justification for study design:
- According to relevant guidelines.
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 03-08-2018 (batch 1) & 18-04-2019 (batch 2).
- Purity test date: 10-08-2016 (batch 1).
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room tempertature.
- Stability under test conditions: Stability for at least 4 days at room temperature and 2 weeks in freezer is confirmed over the concentration range 500 ppm to 15000 ppm, test facility study no.516039.
FORM AS APPLIED IN THE TEST (if different from that of starting material):
-The test item was mixed without use of a vehicle, directly with the required amount of powder feed. - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Han
- Details on species / strain selection:
- Crl: WI(Han) rats were chosen as animal model for this study as it is an accepted rhodent species for toxicity testing by regulatory agencies.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany (female Crl: WI(Han) rats received on 05 Apr 2017, males on 19 Apr 2017).
- Females nulliparous and non-pregnant: yes.
- Age at study initiation: females 13 weeks, males 10 weeks.
- Weight at study initiation: females 204-244 , males 245-289 grams.
- Fasting period before study: no.
- Housing: Macrolon cages, height 18 cm. On arrival and following the pretest (females only) and premating period, 5/cage, MIV type. Mating phase: males and females were cohabitated on a 1:1 basis (MIII type cages). Post-mating phase, males in home cage max 5 males/cage. Females individual ly housed in MIII type cages. During lactation: females were individually housed in MIII type cage. Bedding: Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany.
- Diet (e.g. ad libitum): Ad libitum standard powder rodent diet during acclimatisation period, ad libitum prepared diet thereafter (except for 2 hours during motor activity measurements).
- Water (e.g. ad libitum): Ad libitum municipal water (except for 2 hours during motor activity measurements).
- Acclimation period: At least 5 days prior to start of the pretest period (females) or at least 5 days
before the commencement of administration (males).
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 49-71
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: Not specified. - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): At least every 14 days.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSN IFF® Spezialdiäten GmbH, Soest, Germany).
- Storage temperature of food: Diets were prepared and were kept in the freezer (≤-15°C), if not used on the day of preparation. Diets containing test item were stored at room temperature for a maximum of 4 days. Control diet was stored at room temperature for a maximum of 18 days. - Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: a maximum of 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 post coitum.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Samples were tested for concentration, homogeneity and stability. Analyses were performed by GCFID using a validated analytical procedure (test facility study no. 516039).
- Concentration (all groups) results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration.
- Homogeneity (groups 2 and 4) results were considered acceptable if the relative standard deviation (RSD) of concentrations was <= 10%.
- Stability analyses demonstrated that the test item is stable in the diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study. - Duration of treatment / exposure:
- Males: treated for 28 days (including mating period)
Females (including 14 days prior to mating):
- With offspring: 49-63 days
- Without offspring: 41-51 days - Frequency of treatment:
- Daily
- Details on study schedule:
- Not applicable.
- Dose / conc.:
- 0 ppm
- Remarks:
- Intended dose level: 0 mg/kg bw/day
- Dose / conc.:
- 2 500 ppm
- Remarks:
- Intended dose level: 167 mg/kg bw/day
- Dose / conc.:
- 7 500 ppm
- Remarks:
- Intended dose level: 500 mg/kg bw/day
- Dose / conc.:
- 15 000 ppm
- Remarks:
- Intended dose level: 1000 mg/kg bw/day
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: the dose levels were selected based on the results of a 14-day dose range finder with dietary administration of Ylang Ylang I in rats (test facility study no. 516038 and in an attempt to produce graded responses to the test item.
- Justification of route: the oral route of administration via dietary inclusion was selected because this is a possible route of human exposure during manufacture, handling or use of the test item. - Positive control:
- Not applicable
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
- Arena observations were perfomed weekly.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily.
BODY WEIGHT: Yes
- Time schedule for examinations: on first day of administration, and twic weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on the day of necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, Days 1, 4, 8, 11, 15, 18, 22, 25 and 29, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and daily during lactation.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified.
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: collected on day of sceduled necropsy.
- Anaesthetic used for blood collection: yes, isoflurane.
- Animals fasted: yes, overnight.
- How many animals: selected males and females (5/group).
- Parameters checked: White blood cells (WBC), Neutrophil (absolute), Lymphocyte (absolute), Monocyte (absolute), Eosinophil (absolute), Basophil (absolute), Red blood cells, Reticulocyte (absolute), Red Blood Cell Distribution Width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Plateletd, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: collected on day of sceduled necropsy.
- Animals fasted: yes, overnight.
- How many animals: selected males and females (5/group).
- Parameters checked: Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Total protein, Albumin, Total Bilirubin, Total Bilirubin, Bile Acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate and Thyroid hormone.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males were examined during week 4, females were examined during the last week of lactation.
- Dose groups that were examined: selected males and females (5/group).
- Battery of functions tested: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity.
IMMUNOLOGY: No - Oestrous cyclicity (parental animals):
- Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
- Sperm parameters (parental animals):
- Parameters examined in male parental generation: testis weight, epididymis weight.
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in F0 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups, clinical pathology.
GROSS EXAMINATION OF DEAD PUPS:
yes, determinaton of sex internally and externally and external abnormalities.
ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not examined
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not examined - Postmortem examinations (parental animals):
- GROSS PATHOLOGY: Yes, post mortem examination for all animals with special attention to the reproductive organs.
-Organs weighed at necropsy for selected animals (5/group): Brain; Epididymis; Gland, adrenal; Gland, coagulation; Gland, parathyroid; Gland, prostate; Gland, seminal vesicle; Gland, thyroid; Heart; Kidney; Liver; Ovaries; Spleen; Testes; Thymus; Uterus.
-Organs weighed at necropsy for all remaining animals: Epididymis; Gland, coagulation; Gland, parathyroid; Gland, prostate; Gland, seminal vesicle; Gland, thyroid; Testes.
HISTOPATHOLOGY: Yes, basic tissues were collected and preserved for all animals.
-Tissue collection and preservation for selected animals (5/group): Animal identification; Artery, aorta; Body cavity, nasopharynx; Bone marrow; Bone, femur; Bone, sternum; Brain (seven levels); Cervix; Epididymis; Esophagus; Eye; Gland, adrenal; Gland, coagulation; Gland, harderian; Gland, lacrimal; Gland, mammary; Gland, parathyroid; Gland, pituitary; Gland, prostate; Gland, salivary; Gland, seminal vesicle; Gland, thyroid; Gross lesions/masses; Gut-associated lymphoid tissue; Heart; Kidney; Large intestine, cecum; Large intestine, colon; Large intestine, rectum; Larynx; Liver; Lung; Lymph node (mandibular and mesenteric site); Muscle, skeletal; Nerve, optic; Nerve, sciatic; Ovaries; Pancreas; Skin; Small intestine, duodenum; Small intestine, ileum; Small intestine, jejunum; Spinal cord; Spleen; Stomach; Testes; Thymus; Tongue; Trachea; Urinary bladder; Uterus; Vagina.
-Tissue collection and preservation for all remaining animals (incl. females that failed to deliver pups; nonpregnant and female with total litter loss): Animal identification; Cervix; Epididymis; Gland, coagulation; Gland, mammary; Gland, parathyroid; Gland, pituitary; Gland, prostate; Gland, seminal vesicle; Gland, thyroid; Gross lesions/masses; Ovaries; Testes; Uterus; Vagina. - Postmortem examinations (offspring):
- GROSS NECROPSY
- Gross necropsy consisted of body wieghts, sex ratio, macroscopy. - Statistics:
- All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion.
- Parametric: datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
- Non-Parametric: datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
- Incidence: an overall Fisher’s exact test was used to compare all groups at the 5% significance level. - Reproductive indices:
- Mating index, precoital time, number of implantation sites, fertility index, gestation index and duration, post-implantation survival index
- Offspring viability indices:
- Litter size, live birth index, viability index, lactation index
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Piloerection was noted on a single occasion at the end of lactation for one control female (no. 50), three 7500 ppm females (nos. 62, 68 and 69) and four 15000 ppm females (nos. 73, 76, 79 and 80). For one 15000 ppm female (no. 78) piloerection was noted on three consecutive days during the first week of lactation. These findings where considered incidental and therefore not treatment related.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Description (incidence):
- Female no. 70 (7500 ppm) was euthanized on PND 4 due to total litter loss.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- No treatment-related changes in body weight or body weight gain were observed in males up to 7500 ppm and in females at 2500 ppm.
In males, body weight and body weight gain was significantly decreased for males at 15000 ppm from Day 4 of the pre-mating phase to Day 15 of the mating period. In females, the following effects were observed:
- At 15000 ppm showed a body weight loss Days 1-11 of the pre mating period. At 15000 ppm differences in body weight gain of -6% for males on Day 15 of the mating period and -5% for females on Day 1 of the mating period were noted, compared to controls.
- In the post-coitum phase, body weights for females treated with 7500 ppm and 15000 ppm were lower compared to controls from Day 0 onwards, which was statistically significant on Day 17 (-7%) for 7500 ppm and 15000 ppm and on Day 20 (-10 %) for 7500 ppm only.
- During lactation, bodyweights for females treated with 7500 ppm and 15000 ppm were lower compared to controls. This was statistically significant for females treated with 15000 ppm during the whole lactation period. For females treated with 7500 ppm this was statistically significant on Day 4 of lactation. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- No treatment-related or toxicologically relevant changes in food consumption before or after allowance for body weight were observed in males up to 7500 ppm and for females at 2500 ppm. In males, food consumption was reduced for 15000 ppm males compared to controls on Days 1-8 of the premating period (both absolute and relative) and Days 1-15 of the mating period. Food consumption corrected for body weight was reduced for 7500 ppm and 15000 ppm males compared to control on Days 1-8 of the mating period. In females of the 15000 ppm dose group, food consumption (both absolute and relative) was reduced compared to controls on Days 1-8 of the premating period, Days 0-20 during the post coitum period and Days 1-5 and Days 6-14 of lactation. For 7500 ppm females food consumption (both absolute and relative) was significantly reduced on Days 1-14 during lactation, which was statistically significant on several days.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- In males, a statistically significant increase (1.2 fold) in creatinine for males at 15000 ppm compared to control was noted. These findings are considered to be adverse.
In females, an increase in ALP was noted, which was statistically significant at 15000 ppm (3.8-fold increase). Although female no. 76 had a markedly higher ALP value, all other females at 15000 ppm had a higher ALP value compared to controls as well. Other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- In males, a test item-related microscopic findings were noted in the kidney of the 7500 and 15000 ppm treated males (hyaline droplet accumulation increased basophilia and granular casts). The hyaline droplet accumulation was considered to represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This is a male rat specific protein and therefore the finding is not considered relevant to humans. In females, test item-related microscopic findings were noted in the thymus at 15000 ppm (minimal lymphoid depletion).
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Reproductive function: oestrous cycle:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Minor and incidental effects were observed and therefore considered non-treatment-related.
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Minor and incidental effects were observed on number of implantation sites and fertility index but no dose-response was observed and therefore these findings were considered non-treatment-related.
No effects were observed on mating index and precoital time.
There were 2/10 couples of the control group and 4/10 couples treated at 7500 ppm, that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain the reproductive failure in these couples, except for couple 61/21 at 7500 ppm. Male 21 of this couple showed a disturbed spermatogenesis which was visible by a bilateral moderate degeneration/atrophy of germ cells in the testes together with luminal cell debris in the epididymides. All other males did not show any indications for abnormal spermatogenesis after stage aware evaluation of the testes. There was 1/10 couples treated at 7500 ppm with total litter loss. Histopathology did not reveal changes in the reproductive organs that could explain this although a decrease in mammary gland development (compared to normal lactating/pregnant females) may possibly be related to the litter loss. - Key result
- Dose descriptor:
- NOAEL
- Remarks:
- Reproduction
- Effect level:
- 1 301 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Remarks on result:
- other: Highest dose tested
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- Reproduction
- Effect level:
- 1 590 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Remarks on result:
- other: Highest dose tested
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The clinical signs observed incidentally in treated pups remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment. The viability indices were 99, 98, 87 and 100% in control, 2500 ppm, 7500 ppm and 15000 ppm group respectively.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body weights of male and female pups were lower at 15000 ppm from PND 7–13. The difference from controls was approximately 17% on PND 13.
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- Developmental
- Generation:
- F1
- Effect level:
- 718 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- body weight and weight gain
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- Developmental
- Generation:
- F1
- Effect level:
- 953 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- body weight and weight gain
- Key result
- Reproductive effects observed:
- no
- Conclusions:
- Under the conditions of this study, the NOAEL for reproduction was considered to be 15000 ppm corresponding to 1301 mg/kg bw/day in males and 1590 mg/kg bw/day in females. The developmental NOAEL was considered to be 7500 ppm, corresponding to 718 mg/kg bw/day in males and 953 mg/kg bw/day in females. Based on this result, Ylang Ylang I does not have to be classified for reproductive toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Executive summary:
The reproductive and developmental toxicity potential of Ylang Ylang I was tested under GLP in a combined repeated dose toxicity study with reproduction/developmental toxicity screening test according to OECD TG 422. The experiment was performed by oral administration of the test substance via diet with 5 rats per sex per dose. The tested dietary doses corresponded to 0, 2500, 7500 and 15000 ppm. Males were treated for 28 days (a minimum of two weeks prior to mating and during the mating period). Females that delivered offspring were treated 49-63 days, a minimum of two weeks prior to mating. Females that delivered no healthy offspring were treated for 41 -51 days. The following parameters and endpoints were evaluated in this study for repeated dose toxicity: mortality/ moribundity, clinical signs, functional observations and locomotor activity, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4, gross necropsy findings, organ weights and histopathologic examinations. The following parameters were evaluated in this study for reproduction and development: Mating index, preciodal time, number of implantation sites, fertility index, gestation index and duration, post implantation survival index, litter size, live birth index, viability index, lactation index. The pups were evaluated for clinical signs, body weights, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry (T4) and macroscopic changes.
In the parental animals, test item related changes included a dose dependent increase in liver weights (relative to body weight was significant), at all dose levels in males and in females at 15000 ppm, in the absence of morphological alterations. For females, but not males, an increase in ALP was noted which was statistically significant at 15000 ppm (3.8–fold increase) which in the presence of the increased liver weight was considered adverse. This increase in liver weights (>20% compared to control) were considered adverse in males and females at 15000 ppm. Test item related increase in kidney weights (only relative to body weight was significant) were observed in males and females treated at 15000 ppm. For females the increased kidney weight in the absence of morphological changes was considered non adverse. In males morphological alterations consisted of a combination of increased hyaline droplet accumulation, increased basophilia and granular casts in males treated at 7500 ppm and 15000 ppm. In addition, creatinine levels were statistically significantly increased for males treated with 15000 ppm. The hyaline droplet accumulation was considered to represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein and is not considered relevant to humans risk assessment. No mortality occurred throughout the study. One female was euthanized on PND 4 due to total litter loss. No relevant clinical signs or neurotoxicity were observed. In addition, no treatment related effects on reproductive toxicity were observed up to the highest dose. In the offspring, no developmental toxicity was observed up to 7500 ppm. Body weights of pups of the 15000 ppm dose group were statistically significantly reduced on PND 7 and 13 (approximately 17% lower compared to control on PND 13), which was considered adverse.
Under the conditions of this study, the NOAEL for reproduction was considered to be 15000 ppm corresponding to 1301 mg/kg bw/day in males and 1590 mg/kg bw/day in females. The developmental NOAEL was considered to be 7500 ppm, corresponding to 718 mg/kg bw/day in males and 953 mg/kg bw/day in females. Based on this result, Ylang Ylang I does not have to be classified for reproductive toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Referenceopen allclose all
Parental test article intake
| Group no | 2 | 3 | 4 | |
| Nominal dietary inclusion level (ppm) | 2500 | 7500 | 15000 | |
| Intended dose level (mg/kg bw/day) | 167 | 500 | 1000 | |
| Males Test article intake (mg/kg bw/day) |
Pre-mating | 219 (204 - 244) | 650 (611 - 687) | 1175 (995 - 1127 ) |
| post-mating | 257 (190 – 432) | 785 (616 - 1100 ) | 1426 (1094 - 2192) | |
| Mean of means* | 238 | 718 | 1301 | |
| Females Test article intake (mg/kg bw/day) |
Pre-mating | 221 (208 - 245) | 651 (581 - 754) | 1009 (728 - 1166) |
| Post-coitum | 281 (239 - 316) | 832 (710 - 945) | 1223 (1130 - 1370) | |
| Lactation | 540 (295 - 803) | 1449 (667 - 2194) | 2738 (1525 - 3614 ) | |
| Mean of means* | 337 | 953 | 1590 |
*Mean of means of all periods, weighed for number of measurement intervals per period:
Males: ((4x mean premating) + (4x mean mating)) / 8
Females: ((15 x mean premating) + (20 x mean post-coitum) + (14 x mean lactation)) / 49
Parental test article intake
| Group no | 2 | 3 | 4 | |
| Nominal dietary inclusion level (ppm) | 2500 | 7500 | 15000 | |
| Intended dose level (mg/kg bw/day) | 167 | 500 | 1000 | |
| Males Test article intake (mg/kg bw/day) |
Pre-mating | 219 (204 - 244) | 650 (611 - 687) | 1175 (995 - 1127 ) |
| post-mating | 257 (190 – 432) | 785 (616 - 1100 ) | 1426 (1094 - 2192) | |
| Mean of means* | 238 | 718 | 1301 | |
| Females Test article intake (mg/kg bw/day) |
Pre-mating | 221 (208 - 245) | 651 (581 - 754) | 1009 (728 - 1166) |
| Post-coitum | 281 (239 - 316) | 832 (710 - 945) | 1223 (1130 - 1370) | |
| Lactation | 540 (295 - 803) | 1449 (667 - 2194) | 2738 (1525 - 3614 ) | |
| Mean of means* | 337 | 953 | 1590 |
*Mean of means of all periods, weighed for number of measurement intervals per period:
Males: ((4x mean premating) + (4x mean mating)) / 8
Females: ((15 x mean premating) + (20 x mean post-coitum) + (14 x mean lactation)) / 49
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
Additional information
The reproductive and developmental toxicity potential of Ylang Ylang III oil was read across from Ylang Ylang I. Ylang Ylang I was tested under GLP in a combined repeated dose toxicity study with reproduction/developmental toxicity screening test according to OECD TG 422. The experiment was performed by oral administration of the test substance via diet with 5 rats per sex per dose. The tested dietary doses corresponded to 0, 2500, 7500 and 15000 ppm. Males were treated for 28 days (a minimum of two weeks prior to mating and during the mating period). Females that delivered offspring were treated 49-63 days, a minimum of two weeks prior to mating. Females that delivered no healthy offspring were treated for 41 -51 days. The following parameters and endpoints were evaluated in this study for repeated dose toxicity: mortality/ moribundity, clinical signs, functional observations and locomotor activity, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4, gross necropsy findings, organ weights and histopathologic examinations. The following parameters were evaluated in this study for reproduction and development: Mating index, preciodal time, number of implantation sites, fertility index, gestation index and duration, post implantation survival index, litter size, live birth index, viability index, lactation index. The pups were evaluated for clinical signs, body weights, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry (T4) and macroscopic changes.
In the parental animals, test item related changes included a dose dependent increase in liver weights (relative to body weight was significant), at all dose levels in males and in females at 15000 ppm, in the absence of morphological alterations. For females, but not males, an increase in ALP was noted which was statistically significant at 15000 ppm (3.8–fold increase) which in the presence of the increased liver weight was considered adverse. This increase in liver weights (>20% compared to control) were considered adverse in males and females at 15000 ppm. Test item related increase in kidney weights (only relative to body weight was significant) were observed in males and females treated at 15000 ppm. For females the increased kidney weight in the absence of morphological changes was considered non adverse. In males morphological alterations consisted of a combination of increased hyaline droplet accumulation, increased basophilia and granular casts in males treated at 7500 ppm and 15000 ppm. In addition, creatinine levels were statistically significantly increased for males treated with 15000 ppm. The hyaline droplet accumulation was considered to represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein and is not considered relevant to humans risk assessment. No mortality occurred throughout the study. One female was euthanized on PND 4 due to total litter loss. No relevant clinical signs or neurotoxicity were observed. In addition, no treatment related effects on reproductive toxicity were observed up to the highest dose. In the offspring, no developmental toxicity was observed up to 7500 ppm. Body weights of pups of the 15000 ppm dose group were statistically significantly reduced on PND 7 and 13 (approximately 17% lower compared to control on PND 13), which was considered adverse.
Under the conditions of this study, the NOAEL for reproduction was considered to be 15000 ppm corresponding to 1301 mg/kg bw/day in males and 1590 mg/kg bw/day in females. The developmental NOAEL was considered to be 7500 ppm, corresponding to 718 mg/kg bw/day in males and 953 mg/kg bw/day in females.
Effects on developmental toxicity
Description of key information
Developmental toxicity (OECD TG 422): NOAEL 718 mg/kg bw/day in males and 953 mg/kg bw/day in females. (read across from Ylang Ylang I)
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- The full read across justification report is attached under "Attached justification".
23 April 2018 READ-ACROSS STUDY / YLANG YLANG III OIL - REPEATED DOSE TOXICITY AND REPRODUCTIVE TOXICITY I&B9W8768R001F0.1
Executive Summary
According to Annex VIII, 8.6 and 8.7 of the REACh Regulation (EC) No 1907/2006, Repeated dose toxicity and Reproductive toxicity is standard information required for the registration of substances manufactured or imported in quantities of ten tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).
A read-across approach appears appropriate to predict the endpoint “Repeated dose toxicity and Reproductive toxicity” for the substance Ylang Ylang III oil because:
The source substance Ylang Ylang Ext /I /II oil and target substance Ylang Ylang III oil are chemically and in composition very similar and therefore they are expected to exhibit similar properties.
The source substance, gives rise to the highest concern regarding the oral repeated dose toxicity endpoint, as the constituents notified for relevant systemic toxicological potential and/or giving an alert in the QSAR toolbox add up to 8.5 – 70.5 % w/w in the source versus 8.42 – 44.1 % w/w in the target UVCB.This suggests a worst-case situation, implying that the toxicity can be read across to the target substance.
An OECD TG 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, is available for the substance Ylang Ylang I, which has a composition very similar to the target substance.
This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the structures and the Repeated dose toxicity/Reproductive toxicity for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter - Reason / purpose for cross-reference:
- read-across source
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Piloerection was noted on a single occasion at the end of lactation for one control female (no. 50), three 7500 ppm females (nos. 62, 68 and 69) and four 15000 ppm females (nos. 73, 76, 79 and 80). For one 15000 ppm female (no. 78) piloerection was noted on three consecutive days during the first week of lactation. It is not specified if these findings are treatment related.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Female no. 70 (7500 ppm) was euthanized on PND 4 due to total litter loss.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- No treatment-related changes in body weight or body weight gain were observed in females at 2500 ppm. At 15000 ppm a body weight loss was observed on Days 1-11 of the pre-mating period. At 15000 ppm differences in body weight gain of -5% for females on Day 1 of the mating period were noted, compared to controls. In the post-coitum phase, body weights for females treated with 7500 ppm and 15000 ppm were lower compared to controls from Day 0 onwards, which was statistically significant on Day 17 (-7%) for 7500 ppm and 15000 ppm and on Day 20 (-10 %) for 7500 ppm only. During lactation, body weights for females treated with 7500 ppm and 15000 ppm were lower compared to controls. This was statistically significant for females treated with 15000 ppm during the whole lactation period. For females treated with 7500 ppm this was statistically significant on Day 4 of lactation.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- No treatment-related or toxicologically relevant changes in food consumption before or after allowance for body weight were observed in males up to 7500 ppm and for females at 2500 ppm. For 15000 ppm food consumption (both absolute and relative) was reduced compared to controls on Days 1-8 of the premating period, Days 0-20 during the post coitum period and Days 1-5 and Days 6-14 of lactation. For 7500 ppm females food consumption (both absolute and relative) was significantly reduced on Days 1-14 during lactation, which was statistically significant on several days.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- For females an increase in ALP was noted, which was statistically significant at 15000 ppm (3.8-fold increase). Although female no. 76 had a markedly higher ALP value, all other females at 15000 ppm had a higher ALP value compared to controls as well. Other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Immunological findings:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related higher liver and kidney weights were noted in the femals treated at 15000 ppm.
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related microscopic findings were noted in the thymus of females treated at 15000 ppm (minimal lymphoid depletion).
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Number of abortions:
- no effects observed
- Description (incidence and severity):
- Examination of cage debris of pregnant females revealed no signs of abortion.
- Pre- and post-implantation loss:
- no effects observed
- Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- not examined
- Dead fetuses:
- no effects observed
- Description (incidence and severity):
- The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment.
- Changes in pregnancy duration:
- no effects observed
- Description (incidence and severity):
- Examination of cage debris of pregnant females revealed no signs of premature birth.
- Changes in number of pregnant:
- no effects observed
- Other effects:
- not specified
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- reproduction (female)
- Effect level:
- 1 590 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Remarks on result:
- other: Highest dose tested
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body weights of male and female pups were lower at 15000 ppm from PND 7–13. The difference from controls was approximately 17% on PND 13 Body weights of male and female pups were lower at 7500 ppm on PND 13 compared to controls (-9%), since this was within the historical control data of the performing laboratory this was considered not adverse.
- Reduction in number of live offspring:
- no effects observed
- Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- no effects observed
- Changes in postnatal survival:
- no effects observed
- External malformations:
- no effects observed
- Skeletal malformations:
- not examined
- Visceral malformations:
- not examined
- Other effects:
- not specified
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 718 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- fetal/pup body weight changes
- Remarks on result:
- other: based on actual dose F0 males
- Key result
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 1 301 mg/kg bw/day (actual dose received)
- Treatment related:
- yes
- Relation to maternal toxicity:
- not specified
- Dose response relationship:
- not specified
- Relevant for humans:
- yes
- Conclusions:
- The repeated dose toxicity of Ylang Ylang III oil was read across from Ylang Ylang I. Under the conditions of this study, the NOAEL for reproduction was considered to be 15000 ppm corresponding 1590 mg/kg bw/day in females. The developmental NOAEL was considered to be 7500 ppm, corresponding to 718 mg/kg bw/day (worst-case value from F0 males). Based on these results, Ylang Ylang III does not have to be classified for reproductive toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Executive summary:
The repeated dose toxicity of Ylang Ylang III oil was read across from Ylang Ylang I. The reproductive and developmental toxicity potential of Ylang Ylang I was tested under GLP in a combined repeated dose toxicity study with reproduction/developmental toxicity screening test according to OECD TG 422. The experiment was performed by oral administration of the test substance via diet with 5 rats per dose per sex. The tested dietary doses corresponded to 0, 2500, 7500 and 15000 ppm. Males were treated for 28 days (a minimum of two weeks prior to mating and during the mating period). Females that delivered offspring were treated 49-63 days, a minimum of two weeks prior to mating. Females that delivered no healthy offspring were treated for 41 -51 days. The following parameters and endpoints were evaluated in this study for repeated dose toxicity: mortality/ moribundity, clinical signs, functional observations and locomotor activity, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4, gross necropsy findings, organ weights and histopathologic examinations. The following parameters were evaluated in this study for reproduction and development: Mating index, preciodal time, number of implantation sites, fertility index, gestation index and duration, post implantation survival index, litter size, live birth index, viability index, lactation index. The pups were evaluated for clinical signs, body weights, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry (T4) and macroscopic changes.
Test item related changes included a dose dependent increase in liver weights (relative to body weight was significant) in females at 15000 ppm, in the absence of morphological alterations. An increase in ALP was noted which was statistically significant at 15000 ppm (3.8–fold increase) which in the presence of the increased liver weight was considered adverse. This increase in liver weights (>20% compared to control) was considered adverse at 15000 ppm. Test item related increase in kidney weights (only relative to body weight was significant) was observed females treated at 15000 ppm. The increased kidney weight in the absence of morphological changes was considered non adverse. No mortality occurred throughout the study. One female was euthanized on PND 4 due to total litter loss. No relevant clinical signs or neurotoxicity were observed. No reproductive toxicity was observed up to 15000ppm. In the offspring, no developmental toxicity was observed up to 7500 ppm. Treatment related decreased body weights of pups of the 15000 ppm dose group were statistically significantly reduced on PND 7 and 13 (approximately 17% lower compared to control on PND 13), which was considered adverse.
Under the conditions of this study, the NOAEL for reproduction was considered to be 15000 ppm corresponding 1590 mg/kg bw/day in females. The developmental NOAEL was considered to be 7500 ppm, corresponding to 718 mg/kg bw/day (worst-case value from F0 males). The repeated dose toxicity of Ylang Ylang III oil was read across from Ylang Ylang I. Based on these results, Ylang Ylang III does not have to be classified for reproductive toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05-04-2017 to 31-07-2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECDTG 422 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
- Version / remarks:
- 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 03-08-2018 (batch 1) & 18-04-2019 (batch 2).
- Purity test date: 10-08-2016 (batch 1).
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room tempertature.
- Stability under test conditions: Stability for at least 4 days at room temperature and 2 weeks in freezer is confirmed over the concentration range 500 ppm to 15000 ppm, test facility study no.516039.
FORM AS APPLIED IN THE TEST (if different from that of starting material):
-The test item was mixed without use of a vehicle, directly with the required amount of powder feed. - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Han
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany (female Crl: WI(Han) rats received on 05 Apr 2017, males on 19 Apr 2017).
- Females nulliparous and non-pregnant: yes.
- Age at study initiation: females 13 weeks, males 10 weeks.
- Weight at study initiation: females 204-244 , males 245-289 grams.
- Fasting period before study: no.
- Housing: Macrolon cages, height 18 cm. On arrival and following the pretest (females only) and premating period, 5/cage, MIV type. Mating phase: males and females were cohabitated on a 1:1 basis (MIII type cages). Post-mating phase, males in home cage max 5 males/cage. Females individ ual ly housed in MIII type cages. During lactation: females were individually housed in MIII type cage. Bedding: Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany.
- Diet (e.g. ad libitum): Ad libitum standard powder rodent diet during acclimatisation period, ad libitum prepared diet thereafter (except for 2 hours during motor activity measurements).
- Water (e.g. ad libitum): Ad libitum municipal water (except for 2 hours during motor activity measurements).
- Acclimation period: At least 5 days prior to start of the pretest period (females) or at least 5 days before the commencement of administration (males).
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 49-71
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: Not specified. - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): At least every 14 days.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSN IFF® Spezialdiäten GmbH, Soest, Germany).
- Storage temperature of food: Diets were prepared and were kept in the freezer (≤-15°C), if not used on the day of preparation. Diets containing test item were stored at room temperature for a maximum of 4 days. Control diet was stored at room temperature for a maximum of 18 days. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Samples were tested for concentration, homogeneity and stability. Analyses were performed by GCFID using a validated analytical procedure (test facility study no. 516039).
- Concentration (all groups) results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration.
- Homogeneity (groups 2 and 4) results were considered acceptable if the relative standard deviation (RSD) of concentrations was <= 10%.
- Stability analyses demonstrated that the test item is stable in the diet when prepared and stored und er the same conditions at concentrations bracketing those used in the present study. - Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: a maximum of 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 post coitum.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually. - Duration of treatment / exposure:
- Males: treated for 28 days (including mating period)
Females (including 14 days prior to mating):
- With offspring: 49-63 days
- Without offspring: 41-51 days - Frequency of treatment:
- Daily
- Duration of test:
- 28 days
- Dose / conc.:
- 0 ppm
- Remarks:
- Intended dose level: 0 mg/kg bw/day
- Dose / conc.:
- 2 500 ppm
- Remarks:
- Intended dose level: 167 mg/kg bw/day
- Dose / conc.:
- 7 500 ppm
- Remarks:
- Intended dose level: 500 mg/kg bw/day
- Dose / conc.:
- 15 000 ppm
- Remarks:
- Intended dose level: 1000 mg/kg bw/day
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: the dose levels were selected based on the results of a 14-day dose range finder with dietary administration of Ylang Ylang I in rats (test facility study no. 516038 and in an attempt to produce graded responses to the test item.
- Justification of route: the oral route of administration via dietary inclusion was selected because this is a possible route of human exposure during manufacture, handling or use of the test item. - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
- Arena observations were perfomed weekly.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily.
BODY WEIGHT: Yes
- Time schedule for examinations: on first day of administration, and twic weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on the day of necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, Days 1, 4, 8, 11, 15, 18, 22, 25 and 29, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and daily during lactation.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified.
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: collected on day of sceduled necropsy.
- Anaesthetic used for blood collection: yes, isoflurane.
- Animals fasted: yes, overnight.
- How many animals: selected males and females (5/group).
- Parameters checked: White blood cells (WBC), Neutrophil (absolute), Lymphocyte (absolute), Monocyte (absolute), Eosinophil (absolute), Basophil (absolute), Red blood cells, Reticulocyte (absolute), Red Blood Cell Distribution Width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: collected on day of sceduled necropsy.
- Animals fasted: yes, overnight.
- How many animals: selected males and females (5/group).
- Parameters checked: Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Total protein, Albumin, Total Bilirubin, Total Bilirubin, Bile Acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate and Thyroid hormone.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males were examined during week 4, females were examined during the last week of lactation.
- Dose groups that were examined: selected males and females (5/group).
- Battery of functions tested: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity.
GROSS PATHOLOGY: Yes, post mortem examination for all animals with special attention to the
reproductive organs.
-Organs weighed at necropsy for selected animals (5/group): Brain; Epididymis; Gland, adrenal; Gland, coagulation; Gland, parathyroid; Gland, prostate; Gland, thyroid; Heart; Kidney; Liver; Ovaries; Spleen; Thymus; Uterus.
-Organs weighed at necropsy for all remaining animals: Epididymis; Gland, coagulation; Gland, parathyroid; Gland, seminal vesicle; Gland, thyroid.
HISTOPATHOLOGY: Yes, basic tissues were collected and preserved for all animals.
-Tissue collection and preservation for selected animals (5/group): Animal identification; Artery, aorta; Body cavity, nasopharynx; Bone marrow; Bone, femur; Bone, sternum; Brain (seven levels); Cervix; Epididymis; Esophagus; Eye; Gland, adrenal; Gland, coagulation; Gland, harderian; Gland, lacrimal; Gland, mammary; Gland, parathyroid; Gland, pituitary; Gland, prostate; Gland, salivary; Gland, thyroid; Gross lesions/masses; Gut-associated lymphoid tissue; Heart; Kidney; Large intestine, cecum; Large intestine, colon; Large intestine, rectum; Larynx; Liver; Lung;
Lymph node (mandibular and mesenteric site); Muscle, skeletal; Nerve, optic; Nerve, sciatic; Ovaries; Pancreas; Skin; Small intestine, duodenum; Small intestine, ileum; Small intestine, jejunum; Spinal cord; Spleen; Stomach; Thymus; Tongue; Trachea; Urinary bladder; Uterus; Vagina.
-Tissue collection and preservation for all remaining animals (incl. females that failed to deliver pups; nonpregnant and female with total litter loss): Animal identification; Cervix; Epididymis; Gland, coagulation; Gland, mammary; Gland, parathyroid; Gland, pituitary; Gland, prostate; Gland, thyroid; Gross lesions/masses; Ovaries; Uterus; Vagina. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes - Fetal examinations:
- Examinations performed: Mortality, clinical signs, body weight, sex, anogenital distance, aerola/nipple retenten, blood; thyroid hormone (2 pups/litter), necropsy; external examination (all litters)
- Statistics:
- All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion.
- Parametric: datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
- Non-Parametric: datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
- Incidence: an overall Fisher’s exact test was used to compare all groups at the 5% significance level. - Indices:
- Mating index, precoital time, number of implantation sites, fertility index, gestation index and duration, post-implantation survival index, litter size, live birth index, viability index, lactation index.
- Historical control data:
- Historical control data is available from studies performed in the period 2015-2017.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Piloerection was noted on a single occasion at the end of lactation for one control female (no. 50), three 7500 ppm females (nos. 62, 68 and 69) and four 15000 ppm females (nos. 73, 76, 79 and 80). For one 15000 ppm female (no. 78) piloerection was noted on three consecutive days during the first week of lactation. It is not specified if these findings are treatment related.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Female no. 70 (7500 ppm) was euthanized on PND 4 due to total litter loss.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- No treatment-related changes in body weight or body weight gain were observed in females at 2500 ppm. At 15000 ppm a body weight loss was observed on Days 1-11 of the pre-mating period. At 15000 ppm differences in body weight gain of -5% for females on Day 1 of the mating period were noted, compared to controls. In the post-coitum phase, body weights for females treated with 7500 ppm and 15000 ppm were lower compared to controls from Day 0 onwards, which was statistically significant on Day 17 (-7%) for 7500 ppm and 15000 ppm and on Day 20 (-10 %) for 7500 ppm only. During lactation, body weights for females treated with 7500 ppm and 15000 ppm were lower compared to controls. This was statistically significant for females treated with 15000 ppm during the whole lactation period. For females treated with 7500 ppm this was statistically significant on Day 4 of lactation.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- No treatment-related or toxicologically relevant changes in food consumption before or after allowance for body weight were observed in males up to 7500 ppm and for females at 2500 ppm. For 15000 ppm food consumption (both absolute and relative) was reduced compared to controls on Days 1-8 of the premating period, Days 0-20 during the post coitum period and Days 1-5 and Days 6-14 of lactation. For 7500 ppm females food consumption (both absolute and relative) was significantly reduced on Days 1-14 during lactation, which was statistically significant on several days.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- For females an increase in ALP was noted, which was statistically significant at 15000 ppm (3.8-fold increase). Although female no. 76 had a markedly higher ALP value, all other females at 15000 ppm had a higher ALP value compared to controls as well. Other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Immunological findings:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related higher liver and kidney weights were noted in the femals treated at 15000 ppm.
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related microscopic findings were noted in the thymus of females treated at 15000 ppm (minimal lymphoid depletion).
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Number of abortions:
- no effects observed
- Description (incidence and severity):
- Examination of cage debris of pregnant females revealed no signs of abortion.
- Pre- and post-implantation loss:
- no effects observed
- Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- not examined
- Dead fetuses:
- no effects observed
- Description (incidence and severity):
- The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment.
- Changes in pregnancy duration:
- no effects observed
- Description (incidence and severity):
- Examination of cage debris of pregnant females revealed no signs of premature birth.
- Changes in number of pregnant:
- no effects observed
- Other effects:
- not specified
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- reproduction (female)
- Effect level:
- 1 590 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Remarks on result:
- other: Highest dose tested
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body weights of male and female pups were lower at 15000 ppm from PND 7–13. The difference from controls was approximately 17% on PND 13 Body weights of male and female pups were lower at 7500 ppm on PND 13 compared to controls (-9%), since this was within the historical control data of the performing laboratory this was considered not adverse.
- Reduction in number of live offspring:
- no effects observed
- Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- no effects observed
- Changes in postnatal survival:
- no effects observed
- External malformations:
- no effects observed
- Skeletal malformations:
- not examined
- Visceral malformations:
- not examined
- Other effects:
- not specified
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 718 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- fetal/pup body weight changes
- Remarks on result:
- other: based on actual dose F0 males
- Key result
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 1 301 mg/kg bw/day (actual dose received)
- Treatment related:
- yes
- Relation to maternal toxicity:
- not specified
- Dose response relationship:
- not specified
- Relevant for humans:
- yes
- Conclusions:
- Under the conditions of this study, the NOAEL for reproduction was considered to be 15000 ppm corresponding 1590 mg/kg bw/day in females. The developmental NOAEL was considered to be 7500 ppm, corresponding to 718 mg/kg bw/day (worst-case value from F0 males). Based on these results, Ylang Ylang I does not have to be classified for reproductive toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Executive summary:
The reproductive and developmental toxicity potential of Ylang Ylang I was tested under GLP in a combined repeated dose toxicity study with reproduction/developmental toxicity screening test according to OECD TG 422. The experiment was performed by oral administration of the test substance via diet with 5 rats per dose per sex. The tested dietary doses corresponded to 0, 2500, 7500 and 15000 ppm. Males were treated for 28 days (a minimum of two weeks prior to mating and during the mating period). Females that delivered offspring were treated 49-63 days, a minimum of two weeks prior to mating. Females that delivered no healthy offspring were treated for 41 -51 days. The following parameters and endpoints were evaluated in this study for repeated dose toxicity: mortality/ moribundity, clinical signs, functional observations and locomotor activity, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4, gross necropsy findings, organ weights and histopathologic examinations. The following parameters were evaluated in this study for reproduction and development: Mating index, preciodal time, number of implantation sites, fertility index, gestation index and duration, post implantation survival index, litter size, live birth index, viability index, lactation index. The pups were evaluated for clinical signs, body weights, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry (T4) and macroscopic changes.
Test item related changes included a dose dependent increase in liver weights (relative to body weight was significant) in females at 15000 ppm, in the absence of morphological alterations. An increase in ALP was noted which was statistically significant at 15000 ppm (3.8–fold increase) which in the presence of the increased liver weight was considered adverse. This increase in liver weights (>20% compared to control) was considered adverse at 15000 ppm. Test item related increase in kidney weights (only relative to body weight was significant) was observed females treated at 15000 ppm. The increased kidney weight in the absence of morphological changes was considered non adverse. No mortality occurred throughout the study. One female was euthanized on PND 4 due to total litter loss. No relevant clinical signs or neurotoxicity were observed. No reproductive toxicity was observed up to 15000ppm. In the offspring, no developmental toxicity was observed up to 7500 ppm. Treatment related decreased body weights of pups of the 15000 ppm dose group were statistically significantly reduced on PND 7 and 13 (approximately 17% lower compared to control on PND 13), which was considered adverse.
Under the conditions of this study, the NOAEL for reproduction was considered to be 15000 ppm corresponding 1590 mg/kg bw/day in females. The developmental NOAEL was considered to be 7500 ppm, corresponding to 718 mg/kg bw/day (worst-case value from F0 males). Based on these results, Ylang Ylang I does not have to be classified for reproductive toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Referenceopen allclose all
Parental test article intake
| Group no | 2 | 3 | 4 | |
| Nominal dietary inclusion level (ppm) | 2500 | 7500 | 15000 | |
| Intended dose level (mg/kg bw/day) | 167 | 500 | 1000 | |
| Males Test article intake (mg/kg bw/day) |
Pre-mating | 219 (204 - 244) | 650 (611 - 687) | 1175 (995 - 1127 ) |
| post-mating | 257 (190 – 432) | 785 (616 - 1100 ) | 1426 (1094 - 2192) | |
| Mean of means* | 238 | 718 | 1301 | |
| Females Test article intake (mg/kg bw/day) |
Pre-mating | 221 (208 - 245) | 651 (581 - 754) | 1009 (728 - 1166) |
| Post-coitum | 281 (239 - 316) | 832 (710 - 945) | 1223 (1130 - 1370) | |
| Lactation | 540 (295 - 803) | 1449 (667 - 2194) | 2738 (1525 - 3614 ) | |
| Mean of means* | 337 | 953 | 1590 |
*Mean of means of all periods, weighed for number of measurement intervals per period:
Males: ((4x mean premating) + (4x mean mating)) / 8
Females: ((15 x mean premating) + (20 x mean post-coitum) + (14 x mean lactation)) / 49
Parental test article intake
| Group no | 2 | 3 | 4 | |
| Nominal dietary inclusion level (ppm) | 2500 | 7500 | 15000 | |
| Intended dose level (mg/kg bw/day) | 167 | 500 | 1000 | |
| Males Test article intake (mg/kg bw/day) |
Pre-mating | 219 (204 - 244) | 650 (611 - 687) | 1175 (995 - 1127 ) |
| post-mating | 257 (190 – 432) | 785 (616 - 1100 ) | 1426 (1094 - 2192) | |
| Mean of means* | 238 | 718 | 1301 | |
| Females Test article intake (mg/kg bw/day) |
Pre-mating | 221 (208 - 245) | 651 (581 - 754) | 1009 (728 - 1166) |
| Post-coitum | 281 (239 - 316) | 832 (710 - 945) | 1223 (1130 - 1370) | |
| Lactation | 540 (295 - 803) | 1449 (667 - 2194) | 2738 (1525 - 3614 ) | |
| Mean of means* | 337 | 953 | 1590 |
*Mean of means of all periods, weighed for number of measurement intervals per period:
Males: ((4x mean premating) + (4x mean mating)) / 8
Females: ((15 x mean premating) + (20 x mean post-coitum) + (14 x mean lactation)) / 49
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 718 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- Well documented guideline study.
Additional information
The reproductive and developmental toxicity potential of Ylang Ylang III oil was read across from Ylang Ylang I. Ylang Ylang I was tested under GLP in a combined repeated dose toxicity study with reproduction/developmental toxicity screening test according to OECD TG 422. The experiment was performed by oral administration of the test substance via diet with 5 rats per dose per sex. The tested dietary doses corresponded to 0, 2500, 7500 and 15000 ppm. Males were treated for 28 days (a minimum of two weeks prior to mating and during the mating period). Females that delivered offspring were treated 49-63 days, a minimum of two weeks prior to mating. Females that delivered no healthy offspring were treated for 41 -51 days. The following parameters and endpoints were evaluated in this study for repeated dose toxicity: mortality/ moribundity, clinical signs, functional observations and locomotor activity, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4, gross necropsy findings, organ weights and histopathologic examinations. The following parameters were evaluated in this study for reproduction and development: Mating index, preciodal time, number of implantation sites, fertility index, gestation index and duration, post implantation survival index, litter size, live birth index, viability index, lactation index. The pups were evaluated for clinical signs, body weights, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry (T4) and macroscopic changes.
Test item related changes included a dose dependent increase in liver weights (relative to body weight was significant) in females at 15000 ppm, in the absence of morphological alterations. An increase in ALP was noted which was statistically significant at 15000 ppm (3.8–fold increase) which in the presence of the increased liver weight was considered adverse. This increase in liver weights (>20% compared to control) was considered adverse at 15000 ppm. Test item related increase in kidney weights (only relative to body weight was significant) was observed females treated at 15000 ppm. The increased kidney weight in the absence of morphological changes was considered non adverse. No mortality occurred throughout the study. One female was euthanized on PND 4 due to total litter loss. No relevant clinical signs or neurotoxicity were observed. No reproductive toxicity was observed up to 15000ppm. In the offspring, no developmental toxicity was observed up to 7500 ppm. Treatment related decreased body weights of pups of the 15000 ppm dose group were statistically significantly reduced on PND 7 and 13 (approximately 17% lower compared to control on PND 13), which was considered adverse.
Under the conditions of this study, the NOAEL for reproduction was considered to be 15000 ppm corresponding 1590 mg/kg bw/day in females. The developmental NOAEL was considered to be 7500 ppm, corresponding to 718 mg/kg bw/day (worst-case value from F0 males).
Justification for classification or non-classification
Based on the available read-across data the NOAEL for reproduction was considered to be 1301 mg/kg bw/day in males and 1590 mg/kg bw/day in females. The developmental NOAEL was 718 mg/kg bw/day (based on the male F0 dose). Based on these results, Ylang Ylang III does not have to be classified for reproductive toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Additional information
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